For a variety of reasons, there currently exists a gap between the number of targets put forth by therapeutic areas and the number of targets that can be screened in HTS. We will describe how the application of a novel label-free detection platform based upon resonant waveguide gratings can be used to decrease the size of that gap. Unlike technologies which require labels and/or a variety of reagents, by measuring the change in the local index of refraction at the surface of each well of a microplate (manifest as a wavelength shift), the Epic™ System detects the presence of a biomolecular interaction utilizing only the binding partners of interest. A 384-well prototype of the Epic™ System has been used by our researchers and our pharmaceutical partners to study several assay models, including small molecule/small molecule, protein/drug, and protein/protein. The data generated by these assays will be presented.

We present two independent studies focusing on three different GPCR targets utilizing Cellular Dielectric Spectroscopy (CDS) a novel label-free, functional, cell-based assay technology. The first study investigated the complex signal transduction of human MCHR1 expressed heterologously in CHO and U2-OS cells and endogenously in MOLT4 cells. The CDS platform uniquely enabled the dose-dependent differentiation of PTX-sensitive and Ca2+-coupled events simultaneously in the same microplate well and allowed pharmacological analysis of agonist and antagonist compounds. In the second study, CDS was used to perform hit confirmation for both agonists and antagonists with two different GPCR targets. Unique CDS response profiles of the hits were examined to identify non-silent antagonists and non-selective compounds. This allowed more focused pharmacological analyses of the most promising drug candidates. All results were compared to conventional secondary messenger assays. In summary, we simultaneously obtained information on signal transduction and ligand pharmacology without resorting to multiple (varied) assay-formats.

â Galactosidase (â gal) can be genetically divided into two fragments: - Enzyme Acceptor (EA), and a smaller fragment, Enzyme Donor (ED). These two fragments recombine to form competent â gal, via enzyme fragment complementation (EFC). Both EA and ED-fusion proteins can be expressed in mammalian cells, with expression of the former localized to either membrane or nucleus. 'Positional' complementation only occurs, therefore, when the ED: protein fusion translocates, providing homogeneous assays for interrogation of cell pathways resulting in translocation. Trafficking to the cell exterior can also be monitored using fusion proteins expressed in a suitable extracellular loop, flanked in series by consensus protease cleavage sites. Appearance on the cell surface is monitored by addition of a protease and subsequently detected by EFC. Since none of these methods, collectively, employ imaging, they are amenable to high throughput automation systems, enabling researchers to measure intracellular trafficking pathways using luminescence plate readers.